The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

• Keywords
• DLS, cell line, genotoxicity, micronucleus assay, nanomaterials,
• Publication type
• Journal Article MeSH

In the body, engineered nanoparticles (NPs) may be recognized and processed by immune cells, among which macrophages play a crucial role. We evaluated the effects of selected NPs [NM-100 (TiO2), NM-110 (ZnO), NM-200 (SiO2), and NM-300 K (Ag)] on THP-1 macrophage-like cells. The cells were exposed to subcytotoxic concentrations of NPs (1-25 µg/mL) and the expression of immunologically relevant genes (VCAM1, TNFA, CXCL8, ICAM1, CD86, CD192, and IL1B) was analyzed by RT-qPCR. The expression of selected cytokines, growth factors and surface molecules was assessed by flow cytometry or ELISA. Generation of reactive oxygen species and induction of DNA breaks were also analyzed. Exposure to diverse NPs caused substantially different molecular responses. No significant effects were detected for NM-100 treatment. NM-200 induced production of IL-8, a potent attractor and activator of neutrophils, growth factors (VEGF and IGF-1) and superoxide. NM-110 triggered a proinflammatory response, characterized by the activation of transcription factor NF-κB, an enhanced production of proinflammatory cytokines (TNF-α) and chemokines (IL-8). Furthermore, the expression of cell adhesion molecules VCAM-1 and ICAM-1 and hepatocyte growth factor (HGF), as well as superoxide production and DNA breaks, were affected. NM-300 K enhanced IL-8 production and induced DNA breaks, however, it decreased the expression of chemokine receptor (CCR2) and CD86 molecule, indicating potential immunosuppressive activity. The toxicity of ZnO and Ag NPs was probably caused by their intracellular dissolution, as indicated by transmission electron microscopy imaging. The observed effects in macrophages might further influence both innate and adaptive immune responses by promoting neutrophil recruitment via IL-8 release and enhancing the adhesion and stimulation of T cells by VCAM-1 and ICAM-1 expression.

• Keywords
• DNA damage, THP-1 cells, immune response, nanoparticles, reactive oxygen species,
• Publication type
• Journal Article MeSH